Component in the hedgehog signalling pathway

ABSTRACT

The present invention relates to a novel human patched-like gene (PTCH2), which for the first time has been cloned and sequenced. Several alternatively spliced mRNA forms of PTCH2 have been identified, including transcripts lacking segments thought to be involved in sonic hedgehog (SHH) binding and mRNAs with differentially defined 3′ terminal exons. Further, the invention also relates to the protein encoded by the present PTCH2 as well as to functional analogues and variants thereof.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/SE99/01784 which has an Internationalfiling date of Oct. 6, 1999, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to novel molecules such as proteins,polypeptides and nucleotides, involved in the hedgehog signallingpathway with putative involvement in embryonic development andcarcinogenesis. The invention also relates to various novel advantageoususes of the molecules according to the invention, e.g. in diagnosis andtherapy.

BACKGROUND

In the study of the development of cells, fit flies have extensivelybeen used as a model, as they are less complex than mammalian cells.

Pattern formation takes place through a series of logical steps,reiterated many times during the development of an organism. Viewed froma broader evolutionary perspective, across species, the same sort ofreiterative pattern formations are seen. The central dogma of patternformation has been described (Lawrence and Struhl, 1996). Threeinterlocking and overlapping steps are defined. Firstly, positionalinformation in the form of morphogen gradients allocate cells intonon-overlapping sets, each set founding a compartment. Secondly, each ofthese compartments acquire a genetic address, as a result of thefunction of active “selector” genes, that s cell fate within acompartment and also instruct cells and their descendents how tocommunicate with cells in neighboring compartments. The third stepinvolves interactions between cells in adjacent compartments, initiatingnew morphogen gradients, which directly organize the pattern.

Taking these steps in greater detail, one finds the first step inpatterning to be the definition of sets of cells in each primordium.Cells are allocated according to their positions with respect to bothdorsoventral and anterior/posterior axes by morphogen gradients.Allocation of cells in the dorsoventral axis constitutes the germlayers, such as mesoderm or neurectoderm.

In segmentation, the second step (the specification of cell fate in eachcompartment) is carried out by the gene engrailed and elements of thebithorax complex. Engrailed defines anterior and posterior compartmentsboth in segmentation and in limb specification.

The third step in pattern formation, secretion of morphogens, functionsto differentiate patterns within compartments (and thereby establishsegment polarity). Initially, all cells within a compartment areequipotent, but they become diversified to form pattern. Patternformation depends on gradients of morphogens, gradients initiated alongcompartment boundaries. Such gradients are established by a short-rangesignal induced in all the cells of the compartment in which the abovementioned selector gene engrailed is active. For segment polarity, thissignal is Hedgehog. In the adjacent compartment the selector gene isinactive, ensuring that the cells are sensitive to the signal. TheHedgehog signal range is probably only a few rows of cells wide;responding cells become a linear source of a long-range morphogen, thatdiffuses outward in all directions. There are three known Hedgehogs,Sonic (SHH), Indian (IHH) and Desert (DHH). The proteins they encode cansubstitute each for each other, but in wildtype animals, their distinctdistributions result in unique activities. SHH controls the polarity oflimb growth, directs the development of neurons in the ventral neuraltube and patterns somities. IHH controls endochondral bone developmentand DHH is necessary for spermiogenesis. Vertebrate hedgehog genes areexpressed in many other tissues, including the peripheral nervoussystem, brain, lung, liver, kidney, tooth primordia, genitalia andhindgut and foregut endoderm.

Thus, segment polarity genes have been identified in flies as mutations,which change the pattern of structures of the body segments. Mutationsin these genes cause animals to develop the changed patterns on thesurfaces of body segments, the changes affecting the pattern along thehead to tail axis. For example, mutations in the gene patched cause eachbody segment to develop without the normal structures in the center ofeach segment. Instead there is a mirror image of the pattern normallyfound in the anterior segment. Thus, cells in the center of the segmentmake the wrong structures, and point them in the wrong direction withreference to the over all head-to-tail polarity of the animal.

About sixteen genes in the class are known. The encoded proteins includekinases, transcription factors, a cell junction protein, two secretedproteins called wingless (WG) and the above mentioned Hedgehog (HH), asingle transmembrane protein called patched (PTC) and some novelproteins not related to any known protein. All of these proteins arebeleived to work together in signaling pathways that inform cells abouttheir neighbors in order to set cell fates and polarities.

PTC has been proposed as a receptor for HH protein based on geneticexperiments in flies. A model for the relationship is that PTC actsthrough a largely unknown pathway to inactivate both its owntranscription and the transcription of the wingless segment polaritygene. This model proposes that HH protein, secreted from adjacent cells,binds to the PTC receptor, inactivates it and thereby prevents PTC fromturning off its own transcription or that of wingless. A number ofexperiments have shown coordinate events between PTC and HH.

Human patched gene (PTCH) was recently identified as the generesponsible for the nevoid basal cell carcinoma syndrome (NBCCS), alsoknown as the Gorlin Syndrome, which is an autosomal dominant disorderthat predisposes to both cancer and developmental defects (Gorlin (1995)Dermatologic Clinics 13:113-125) characterized by multiple basal cellcarcinomas (BCCs), medulloblastomas and ovarian fibromas as well asnumerous developmental anomalities (Hahn, H., Wicking, C.,Zaphiropoulos, P. G., Gailani, M. R., Shanley, S., Chidambaram, A.,Vorechovsky, I., Holmberg, E., Undén, A. B., Gillies, S., Negus, K.,Smyth, I., Pressman, C., Leffell, D. J., Gerrard, B., Goldstein, A. M.,Dean, M., Toftg{dot over (a)}rd, R., Chenevix-Trench, G., Wainright, B.and Bale, A. E. (1996): “Mutations of the human homolog of Drosophilapatched in the nevoid basal cell carcinoma syndrome”, Cell 85, 841-851;and Johnson, R. L., Rothman, A. L., Xie, J., Goodrich, L. V., Bare, J.W., Bonifas, J. M., Quinn, A: G., Myers, R. M., Cox, D. R., Epstein, E.H. Jr and Scott, M. P. (1996): “Human homolog of patched, a candidategene for the basal cell nevus syndrome”, Science 272, 1668-1671). PTCHcodes for a membrane receptor of the autolytically cleaved (proteinspliced), amino terminal domain of sonic hedgehog (SHH) (Mariago, V.,Davey, R. A., Zuo, Y., Cunningham, J. M. and Tabin, C. J. (1996):“Biochemical evidence that patched is the Hedgehog receptor”, Nature384, 176-179; and Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A.,Gu, Q., Johnson, R. L., Scott, M. P., Pennica, D., Goddard, A.,Phillips, H., Noll, M., Hooper, J. E., de Sauvage, F. and Rosenthal, A.(1996): “The tumor-suppressor gene patched encodes a candidate receptorfor Sonic hedgehog”, Nature 384, 129-134). In the non-signalling state,PTCH is thought to inhibit the consecutive signalling of anothermembrane protein, smoothened (SMO), however binding of SHH to PTCHreleives this inhibition (Goodrich, L. V., Milenkovic, L., Higgins, K.M. and Scott, M. P. (1997): “Altered neural cell fates andmedullablastom in mouse patched mutants”, Science 277, 1109-1113). Thiscascade of signalling events, best characterized in Drsophila, alsoinvolves a number of intracellular components including fused (a serinethreonine kinase), suppressor of fused, costal 2, and cubitusinterruptus (Ruiz i Altaba, A.,: “Catching a Gli-mpse of Hedgehog”(1997) Cell 90, 193-196). The latter is a transcription factor thatpositively regulates the expression of target genes which also includePTCH itself.

Mutations in the PTCH gene have been identified in both sporadic andfamilial BCCs (Gailani, M. R., Stähle-Bäckdahl M., Leffell, D. J.,Glynn, M., Zaphiropoulos, P. G., Pressman, C., Undén, A. B., Dean, M.,Brash, D. E., Bale, A. E. and Toftg{dot over (a)}rd, R. (1996): “Therole of human homologue of Drosophila patched in sporadic basal cellcarcinomas” Nature Gene 14, 78-81). The lack of the normal PTCH proteinin these cells allows the constitutive signalling of SMO to occur,resulting in the accumulation of mutant PTCH mRNAs (Undén, B. A.,Zaphiropolous, P. G., Bruce, K., Toftg{dot over (a)}rd, R., and St{dotover (a)}hle-Bäckdahl M. (1997): “Human patched (PTCH) mRNA isoverexpressed consistently in tumor cells of both familial and sporadicbasal cell carcinoma”, Cancer Res. 57, 2336-2340). WO 96/11260 disclosesthe isolation of patched genes and the use of the PTC protein toidentify ligands, other than the established ligand Hedgehog, that bindthereto.

However, there is still a need of a further understanding of theSHH/PTCH cell signalling, which may be provided by disclosure of furthergenes, peptides and proteins involved therein.

SUMMARY OF THE INVENTIONS

The present invention provides a significant step forward regarding theunderstanding of the above described pathway. By a combination of cDNAlibrary and RACE analysis a novel human patched-like gene (PTCH2) hasbeen cloned and sequenced. Several alternatively spliced mRNA forms ofPTCH2 have been ideintified, including transcipts lacking segmentsthought to be involved in sonic hedgehog (SHH) binding and mRNAs withdifferentially defined 3′ terminal exons. Accordingly, the inventionrelates to isolated such mRNAs as well as to cDNAs complementarythereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genomic sequence of SEQ ID NO:5, wherein exons andintrons are designated in the genomic sequence of the novel humanpatched 2 gene.

FIG. 2A discloses an amino acid sequence comparison of the human PTCH2(residues 1-633 of SEQ ID NO:1)(upper lines) and PTCH1(residues 1-699 ofSEQ ID NO:6) (lower lines) sequences.

FIG. 2B is a representation of the alternative splicing events (SEQ IDNOS:7, 8, 9, 10, 11, 12, 13, 14, 15 and 16) that result in differentC-termini.

FIG. 2C is a representation of the different variations of splicedtranscipts encompassing exon 1 and exon 2 sequences.

FIG. 3A is a dark-field photomicrograph of a BCC tumor hybridised with³⁵S-labeled antisense probe showing abundant signal for PTCH1 mRNA(light grains) in all BCC tumor cells.

FIG. 3B discloses PTCH2 mRNA overexpression in BCC and is in contrastmainly expressed in the basaloid cells in the periphery of the tumornests.

FIG. 3C is another BCC showing a strong PTCH2 mRNA signal in theperiphery of the tumor nest (Tu), wheras no signal is detected inepidermis (Ep).

FIG. 3D are sections of the same tumor (C) hybridised with the PTCH2sense probe showed no signal.

FIG. 3E shows immunoreactivity for Ki-67.

FIG. 3F discloses how tumor nests under high power magnificationdemonstrate abundant PTCH2 mRNA signal (black grains) in the darkbasaloid tumor cells and lower signal in the center (arrow).

DEFINITIONS

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The terms “isolated” “purified” or “biologically pure” refer to materialwhich is substantially or essentially free from components whichnormally accompany it as found in its native state.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical or chemical means. For example, usefullabels include ³²P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as sommonly used in a ELISA), biotin, dioxigenin, or haptens andproteins for which antisera or monoclonal antibodies are available(e.g., the peptide of SEQ ID NO:1 can be made detectable, e.g., byincorporating a radio-label into the peptide, and used to detectantibodies specifically reactive with the peptide).

As used herein a “nucleic acid probe” is defined as a nucleic acidcapable of binding to a target nucleic acid of complementary sequencethrough one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (i.e. A, G, C, or T) ormodified bases (7-deazaguanosine, inosine, etc.) In addition, the basesin a probe may be joined by a linkage other than a phosphodiester bond,so long as it does not interfere with hybridisation. Thus, for example,probes may be peptide nucleic acids in which the constituent bases arejoined by peptide bonds rather that phosphodiester linkages. It will beunderstood by one of skill in the art that probes may bind targetsequences lacking complete complementarity with the probe sequencedepending upon the stringency of the hybridisation conditions. The,probes are preferably directly labeled as with isotopes, chromophores,lumiphore, chromogens, or indirectly labeled such as with biotin towhich a streptavidin complex may later bind. By assaying for thepresence or absence of the probe, one can detect the presence or absenceof the selct sequence or subsequence.

A “labeled nucleic acid probe” is a nucleic acid probe that is bound,either covalently, through a linker, or through ionic, van der Waals orhydrogen bonds to a label such that the presence of the probe may bedetected by detecting the presence of the label bound to the probe.

The term “target nucleic acid” refers to a nucleic acid (often derivedfrom a biological sample), to which a nucelic acid probe is designed tospecifically hybridise. It is either the presence or absence of thetarget nucleic acid that is to be detected, or the amount of the targetnucleic acid that is to be quantified. The target nucleic acid has asequence that is complementary to the nucleic acid sequence of thecorresponding probe directed to the target. The term target nucleic acidmay refer to the specific subsequence of a larger nucleic acid to whichthe probe is directed or to the ovarall sequence (e.g., gene or mRNA)whose expression level it is desired to detect. The difference in usagewill be apparent from context.

The term “recombinant” when used with reference to a cell, or nucleicacid, or vector, indices that the cell, or nucleic acid, or vector, hasbeen modified by the introduction of a heterologous nucleic acid or thealteration of a native nucleic acid, or that the cell is derived from acell so modified.

The term “identical” in the context of two nucleic acids or polypeptidesequences refers to the residues in the two sequences which are the samewhen aligned for maximum correspondence. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorith ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. USA 85: 2444, by computerized implementations of thesealgorithms (GAP, GESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.) or by inspection. The BLAST algorithm performs a statisticalanalysis of the similarity between two sequences; see e.g., Karlin andAltschul (1993) Proc. Nat'l Acad. Sci. USA 90: 5873-5787.

The term “substantial identity” or “substantial similarity” in thecontext of a polypeptide indicates that a polypeptides comprises asequence with at least 70% sequence identity to a reference sequence, orpreferably 80%, or more preferably 85% sequence identity to thereference sequence, or most preferably 90% identity over a comparisonwindow of about 10-20 amino acid residues. An indication that twopolypeptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a polypeptide is substantially identical to a secondpolypeptide, for example, where the two peptides differ only by aconservative substitution.

An indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide which the first nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid. Another indication that two nucleic acid sequencesare substantially identical is that the two molecules hybridise to eachother under stringent conditions.

The phrase “hybridising specifically to”, refers to the binding,duplexing, or hybridising of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. The term “stringentconditions” refers to conditions under which a probe will hybridise toits target subsequence, but to no other sequences. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridise specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point™ for the specific sequence at a definedionic strength and pH. The Tm is the temperature (under defined ionicstrength, pH, and nucleic acid concentration) at which 50% of the probescomplementary to the target sequence hybridise to the target sequence atequilibrium. (As the target sequences are generally present in excess,at Tm, 50% of the probes are occupies at equilibrium). Typically,stringent conditions will be those in which the salt concentration isless than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ionconcentration (or other salts) at pH 7.0 to 8.3 and the temperature isat least about 30° C. for whort probes (e.g., 10 to 50 nucleotides) andat least about 60° C. for long probes (e.g., greater than 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

The term “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The term “immunoassay” is an assay that utilizes an antibody tospecifically bind an analyte. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the analyte.

The phrases “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind preferentially to a particular protein and do not bind in asignificant amount to other proteins present in the sample. Specificbinding to a protein under such conditions requires an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antoibodiesspecifically immunoreactive with a protein. See harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbour Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

A “gene product”, as used herein, refers to a nucleic acid whosepresence, absence, quantity, or nucleic acid sequence is indicative of apresence, absence, quantity, or nucleic acid composition of the gene.Gene products thus include, but are not limited to, and mRNA transcripta cDNA reverse transcribed from an mRNA, and RNA transcribed from thatcDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA or subsequences of any of these nucleic acids.Polypeptides expressed by the gene or subsequences thereof are also geneproducts. The particular type of gene product will be evident from thecontext of the usage of the term.

A “modified drug” means a compound, which retains the pharmaceuticalproperties of the original drug or active substance while the structurethereof has been modified. Further, encompassed by the term “drug” arealso compounds useful in diagnostic methods by their specific bindingproperties.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to an isolated humanprotein, or an analogue or a variant thereof, capable of participatingin the human PTCH/SHH pathway during embryonic development and/orcarcinogenesis, such as basal cell carcinoma. The novel proteinaccording to the invention is encoded by a novel gene, which isolatednucleic acid is described in detail below and which is denoted patched 2(PTCH2) due to its similarities with patched 1 (PTCH1). Accordingly, theprotein according to the invention exhibits substantial differences insequence and functions when compared to human PTCH1 protein. The proteinaccording to the invention is best characterized by its functions whichwhen compared to human PTCH1 are similar but distinct therefrom incertain ways, more specifically disclosed below in the section “Resultsand discussion”. The novel human PTCH2 protein according to theinvention is also distinct from the previously isolated mouse PTCH2.Thus, in the preferred embodiment thereof it comprises a substantialpart of the amino acid sequence disclosed in SEQ ID NO: 1 and submittedto the GenBank under protein id no AAD17260.1. even though it is to beunderstood that the present invention encompasses any fragment, analogueor variant thereof exhibiting the biological functions of the PTCH2protein disclosed herein. Thus, preferably, the present proteincomprises at least about 1000, more preferably at least about 1040 andmost preferably essentially all of the amino acids of the sequencedenoted SEQ ID NO: 1, such as about 1100.

The proteins according to the invention are easily prepared by someoneskilled in this field by recombinant DNA techniques using the moleculesdisclosed below or any synthetic method (see e.g. Barany and Merrifield,Solid-Phase Peptide synthesis, pp. 3-284 in The Peptides: Analysis,Synthesis, Biology, Vol. 2: Special Methods in Peptide synthesis, PartA, Merrifield et al., J. Am. Chem. Soc., 2149-2156).

The present invention also relates to the use of the peptides,polypeptides and proteins disclosed herein as lead compounds in methodsaimed at finding novel substances, i.e. modified drugs, such assubstances exhibiting equivalent or even more advantageous propertiesthan the lead compounds as such. Such modified drugs may also bedesigned by methods of combinatorial chemistry, wherein a structurallysimilar compound is specifically designed e.g. by aid of computers.Alternatively, the present modified drug is identidied by screening of alibrary of candidate compounds, e.g. using an antibody according to theinvention. In the present context it is to be understood that when sucha modified drug has been identified, it is possible to produce it by anyother suitable technique. The invention also relates to proteomicmethods wherein the present molecules are used as well as to such a useper se.

A second aspect of the present invention is a nucleic acid encoding aprotein, an analogue or a variant thereof as defined above, that is, theprotein coding region of the novel human isolated PTCH2 gene. The PTCH2gene is 57% identical to PTCH1 and 91% identical to the published mousePtch2 sequence (see Motoyama et al., (1998), supra). Thus, preferably,the nucleic acid according to the present invention comprises at leastabout 3000 bases, more preferably at least about 3094 bases and mostpreferably essentially all of the sequence denoted SEQ ID NO: 2.

In a specific aspect, the present invention relates to the isolatedhuman genomic PTCH2 nucleic acid comprising parts or all of the genomicsequence denoted SEQ ID NO: 5. In the disclosure of the genomic sequenceshown in FIG. 1, the exon/intron structure of the present gene is shown.Further to the exons shown therein, exon 12 a and 12 b has also beenidentified, as specifically defined by SEQ ID NO:3 and SEQ ID NO:4,respectively. Interestingly, there is a splice variant that joins exon12 a to a 3′ segment of exon 12 b with conservation of the intronicGT-AG dinucleotides. Exons 12 a and 12 b are not variants, but theactual exons of the gene identified by sequencing the correspondinggenomic region. (Materials and methods were as discribed beloow).Accordingly, these findings show that PTCH2 has the same intron/exonstructure organization as PTCH1. In another embodiment of this aspect,the present invention relates to a transcript that has skipped only oneof the exons 9 and 10 defined in FIG. 1. In an alternative embodiment,the transcript according to the invention has skipped both of exon 9 and10. The splice variants of the present gene are discussed in more detailbelow in the section “Results”, all of which are included within thescope of the present invention. This aspect of the inventionadvantageously enables design of suitable PCR primers, which in turnenables screening for mutations of all of the coding sections thereof,e.g. by SSCP analysis, sequencing, or any other suitable method known tosomeone skilled in this field. Thus, the novel human PTCH2 geneaccording to the invention has been localized by radiation hybridmapping to chromosome 1p32-35 with D1S211 and WI-1404 as closestflanking markers and with an estimated localization 5.5cR from D1S443.This region is often lost by LOH in various different tumor types, suchas neuroblastoma, melanoma, breast cancer, colon cancer etc.Accordingly, PTCH2 is a candidate for a tumor suppressor gene in thisregion and the present invention also encompass diagnostic methods basedon this new disclosure.

To this chromosomal region, three cancer predisposition syndromes havealso been mapped, namely, familial melanoma CMM1, modifier locus forfamilial adenomatous polyposis hMom1 and Michelin Tire Baby Syndrome.PTCH2 is further a candidate for the gene behind these heritarysyndromes. The present molecules are therefore advantageously used inthe context of these conditions, e.g. in therapy and/or diagnosis, suchas in assays.

Further, the invention also relates to various PCR primers based onintronic sequences, allowing amplification of all coding sequence. Suchprimers are advantageously used for mutation screening.

Further, the present invention also relates to the any isolated nucleicacid capable of specifically hybridising to a nucleic acid according tothe invention. In addition, the invention also relates to such anisolated nucleic acid which comprises one o more mutations compared tothe genomic sequence as well as the use of the novel isolated nucleicacids, e.g. to identify mutations for diagnostic and/or therapeuticpurposes.

Further embodiments of this aspect of the invention includes nucleicacid probes, e.g. DNA probes, labelled nucleic acids, cDNAs, RNAs etc.,that is, all gene products obtainable by someone skilled in this fieldbased on the novel isolated human PTCH2 gene.

Another aspect of the invention is a nucleic acid corresponding to anyone of the splicing variants disclosed in FIG. 2B, a protein orpolypeptide encoded thereof as well as various uses thereof.

As regards the preparation of nucleic acids according to the invention,any suitable recombinant DNA technique or synthetic method may be used.(For general laboratory procedures useful in this context, see e.g.Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed.,Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol. 153, Academic Press, Inc., San Diego, Calif.; CurrentProtocols in Molecular Biology, F. M. Ausbel et al., eds., CurrentProtocols (1994)).

A further aspect of the present invention is a vector comprising anucleic acid as defined above. Vectors are e.g. useful for transformingcells in vitro or in vivo to express the proteins and peptides accordingto the invention and may e.g be plasmids, viruses etc.

Another aspect of the invention is a recombinant cell such as aeucaryotic, e.g. a mammalian cell, or a procaxyotic cell, e.g. abacteria, comprising a vector as defined above. Such cells may e.g. beused to monitor expression levels of the proteins and polypeptidesaccording to the invention in a wide variety of contexts. For example,when the effects of a drug is to be determined, the drug will beadministered to the transformed organism, tissue or cell. Accordingly,model systems including such cells are another aspect of the invention.

A further aspect of the invention is an antibody, such as a monoclonalor polyclonal antibody, which specifically binds to a protein orpolypeptide according to the invention. An exemplary imunuoglobulin(antibody) structural unit comprises a tetramer. Each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). TheN-terminus of each chain defines a variable region of about 100 to 110or more amino acids primarily responsible for antigen recognition. Theterms variable light chain (V_(L)) and variable haeavy chain (V_(H))refer to these light and heavy chains, respectively.

The invention also encompasses chimeric or other antibodies that bindsthe present proteins or polypeptides. Further, the invention alsorelates to the use of the present antibodies in assays. (In thiscontext, see e.g. Fundamental Immunology, Third Edition, W. E. Paul,ed., Raven Press, N.Y. 1993).

Further, the invention also relates to a recombinant cell expressing anantibody according to the invention.

In general, prokaryotes can be used for cloning the DNA sequencesencoding a human anti-PTCH2 immunoglobulin chain. E. coli is oneprokaryotic host particularly useful for cloning the DNA sequences ofthe present invention. Microbes, such as yeast are also useful forexpression Saccharomyces is a preferred yeast host, with suitablevectors having expression control sequences, an origin of replication,termination sequences and the like as desired. Typical promoters include3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters include, among others, promoters from alcohol dehydrogenase 2,isocytochrome C, and enzymes responsible for maltose and galactoseutlization.

Mammalian cells are a particularly preferred host for expressingnucleotide segments encoding immunoglobulins or fragments thereof (see,e.g. Winnacker, From Genes to Clones, VCH Publishers, N.Y., 1987). Anumber of suitable host cell lines cable of secreting intactheterologous proteins have been developed in the art and include CHOcell lines, various COS cell lines, HeLa cells, L cells and myeloma celllines. Preferably, the cells arm nonhuman. Expression vectors for thesecells can include expression control sequences, such as an origin ofreplication, a promoter, an enhancer (Queen et al. (1986) Immunol. Rev.89:49), and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from endogenous genes, cytomegalovirus,SV40, adenovirus, bovine pappillomavirus, and the like (see, e.g., Co etal. (1992) J. Immunol. 1458: 1149).

An additional aspect of the present invention is a kit for the detectionof a human PTCH2 gene or polypeptide comprising in a container amolecule selected from the group consisting of a nucleic acid, apolypeptide or a protein or an antibody according to the invention.Further suitable components of such a kit are easily determined bysomeone skilled in this field as are the conditions for the use thereof.

Further, the invention also realtes to the use of a nucleic acidselected from the group consisting of SEQ ID NOS: 2-4 and SEQ ID NO: 5in gene therapy. In addition to said specifically disclosed sequences,any one of the herein disclosed exons may be used to this end. For areview of gene therapy procedures, see Anderson, Science (1992)256:808-813; Nabel and Felgner (1993) TIBTECH 11: 211-217; Mitani andCaskey (1993) TIBTECH 11: 162-166; Mulligan (1993) Science 926-932;Dillon (1993) TIBTECH 11: 167-175; Miller (1992) Nature 357: 455-460;Van Brunt (1988) Biotechnology 6(10): 1149-1154; Vigne (1995)Restorative Neurology and Neuroscience 8: 35-36; Kremer and Perricaudet(1995) British Medical Bulletin 51(1) 31-44; Haddada et al. (1995) inCurrent Topics in Microbiology and Immunology Doerfler and Böhm (eds)Springer-Verlag, Heidelberg Germany; and Yu et al., Gene Theraphy(1994)1:13-26.

Delivery of the gene or genetic material into the cell is the firstcritical step in gene therapy treatment of disease. A large number ofdelivery methods are well known to those of skill in the art. Suchmethods include, for example liposome-based gene delivery (Debs and Zhu(1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques6(7): 682-691; Rose U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309;and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414), andreplication-defective retroviral vectors harboring a therapeuticpolynucleotide uence as part of the retroviral genome (see, e.g., Milleret al. (1990) Mol. Cell. Biol. 10:4239 (1990; Kolberg (1992) J. NIH Res.4:43, and Cornetta et al. Hum. Gene Ther. 2:215 (1991)). Widely usedretroviral vectors include those based upon nurine leukemia virus(MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus(SIV), human immuno deficiency virus (HIV), and combinations thereofSee, e.g., Buchscher et al. (1992) J. Virol. 66 (5) 2731-2739; Johann etal. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt et al., (1990)Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller etal., J. Virol. 65:2220-2224 (1991); Wong-Staal et al., PCT/US94/05700,and Rosenburg and Fauci (1993) in Fundamental Immunology, Third EditionPaul (ed) Raven Press, Ltd., New York and the references therein, and Yuet al., Gene Therapy (1994) supra).

The present invention may also be used in the pharmaceutical industry.For example, it will provide information that eventually may enablecells from fetal tissue, which may the be transplanted into patientssuffering from e.g. Parkinson's disease or cancer, such as BCC. (For abrief review of methods of drug delivery, see Langer 249:1 527-1533(1990), Remington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17^(th) ed. (1985) etc.)

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genomic sequence of SEQ ID NO:5, wherein exons andintrons are designated in the genomic sequence of the present humanpatched 2 gene. However, exons 12 a and 12 b discussed above are notspecifically shown in FIG. 1, but is instead disclosed as the separatesequences SEQ ID NO:3 and SEQ ID NO:4, respectively. FIG. 2A disclosesan amino acid sequence comparison of the human PTCH2(residues 1-633 ofSEQ ID NO:1)(upper lines) and PTCH1(residues 1-699 of SEQ ID NO:6)(lower lines) sequences. Vertical lines indicate identical amino acids,while dots similar amino acids. The PTCH2 sequence presented is composedof the original cDNA clones and of the products of the 5′ RACE analysis.

FIG. 2B is a representation of the alternative splicing events (SEQ IDNOS:7, 8, 9, 10, 11, 12, 13, 14, 15 and 16) that result in differentC-termini. In the parotid gland and the colon, the penultimate and thelast exon are canonically joined together. In fetal brain however thepenultimate exon with part of the 3′ intron functions as the terminalexon. The intronic sequence is shown by small letters with the flankingexonic by capital letters. Above the nucleotide sequence, the deducedamino acid sequence is shown, and below is the corresponding sequence ofthe mouse Ptch2. The conserved intronic dinucleotides are shown by boldletters and the termination signals are indicated by asterisks. Note theabsence of conservation of the position of the termination codonsbetween the mouse and human PTCH2 sequences. The putativepolyadenylation signals are also shown in this diagram. The genomicorganization was obtained by analyzing BAC clones encompassing the PTCH2gene.

FIG. 2C is a representation of the different variations of splicedtranscipts encompassing exon 1 and exon 2 sequences. The canonical exons1 and 2 are shown by boxes and the intron between them by a solid line.The GT and AG dinucleotides spanning the sequences that are used asintrons in individual transcripts are indicated by small letters. G,Genomic structure, derived from sequencing segements of BAC clonesencompassing the PTCH2 gene; C, Canonical transcript; A, Transcript A(the skipped exons 9 and 10 of this product are not shown in thediagram); B, Transcript B.

FIG. 3A is a dark-field photomicrograph of a BCC tumor hybridised with³⁵S-labeled antisense probe showing abundant signal for PTCH1 mRNA(light grains) in all BCC tumor cells.

FIG. 3B discloses PTCH2 mRNA overexpression in BCC and is in contrastmainly expressed in the basaloid cells in the periphery of the tumornests.

FIG. 3C is another BCC showing a strong PTCH2 mRNA signal in theperiphery of the tumor nest (Tu), wheras no signal is detected inepidermis (Ep).

FIG. 3D are sections of the same tumor (C) hybridised with the PTCH2sense probe showed no signal.

FIG. 3E shows immunoreactivity for Ki-67 (brown precipitate) seen in theperiphery, in the cells that showed strong upregulation of PTCH2 mRNA.

FIG. 3F discloses tumor nests under high power magnification demonstrateabundant PATCH2 mRNA signal (black grains) in the dark basaloid tumorcells and lower signal in the center (arrow). Bars (A-E), 24 μm, and F,6 μm.

EXPERIMENTAL Materials and Methods

In the present context, a general reference is made to G. Zaphiropouloset al., Cancer Res., vol. 59, p. 787-792, Feb. 15, 1999, disclosinguseful methods in the present context. All references mentioned in thepresent application are hereby included herein by reference. Theexamples below are not intended to limit the scope of the invention butmerely as an illusion.

The RACE analysis was performed essentially as described before(Zaphiropoulos, P. G. and Toftg{dot over (a)}rd, R. (1996): “cDNAcloning of a novel WD repeat protein mapping to the 9q22.3 chromosomalregion”, DNA Cell Biol. 15, 1049-1056) using the Marathon kit (Promega).The primer sequences used for RACE are available upon request.

The PTCH2, 35S-labeled RNA probes used for the in situ hybridisations,that were performed as previously described (Undén et al., (1997),supra), corresponded to positions 218 to 437 and 838 to 920 in the PTCH2sequence of SEQ ID NO:1.

Results and Discussion

In order to identify additional components of the PTCH(SHH cascade ofsignalling events, the Incyte LifeSeq™ database (Incyte PharmaceuticalsInc., Palo Alto, Calif., USA) was searched using PTCH sequences. Inaddition to clones representing the PTCH cDNA, two nearly identicalcDNAs were identified, from the parotid gland and the colon, thatcontained sequences similar to, but distinct from, the 3′ end of PTCH.By 5′ RACE analysis using fetal brain cDNAs additional sequenceinformation from these transcripts (termed PTCH2) and corresponding to afill length cDNA, was obtained (FIG. 2A ). PTCH2 is 57% identical toPTCH1, with a significantly variable region present between thetransmembrane domains 6 and 7, and 91% identical to the recentlypublished mouse Ptch2 sequence (Motoyama, J., Takabatake, T., Takeshina,K. and Hui, C. (1998): “Ptch2, a second mouse Patched gene isco-expressed with Sonic hedgehog”, Nature Genet. 18, 104-106). Insimilarity with the mouse gene, PTCH2 lacks the C-terminal extensionpresent in human, mouse and chicken PTCH1 (Goodrich, L. V., Johnson, R.L., Milenkovic, L., McMahon, J. A., and Scott, M. P. (1996):“Conservation of the hedgehog/patched signalling pathway from flies tomice: Induction of a mouse patched gene by Hedgehog”, Genes Dev. 10,301-312, Marigo, V., Scott, M. P., Johnson, R. L., Goodrich, L. V. andTabin, C. J. (1996): “Conservation in hedgehog signalling: Induction ofa chicken patched homolog by Sonic hedgehog in the developing limb”,Development 122, 1225-1233). However, according to the presentinvention, it has been shown that the human PTCH2 cDNA terminates 36amino acids earlier that the mouse Ptch2 sequence. Moreover, when 3′RACE was perfomed from fetal brain, an alternate C-terminal region wasidentified. This had a high structural similarity with the mouse Ptch2C-terminal sequence and originates from the genomic region that linksthe last two exons of PTCH2 (FIG. 2B). Therefore, in these alternativelyspliced transcripts, the penultimate exon with a segment of thecontiguous 3′ intron serves as the terminal exon.

Moreover the human and mouse transcripts differed in the position of thetermination signals (the human sequence is 21 amino acids longer),suggesting a non-conserved, species-specific function of this alternateC-terminal domain. The finding of two possible C-terminal regions forPTCH2 is intriguing and implies a role of this phenomenon in modulatingsignalling. Additional alternatively spliced transcripts were alsoidentified by the RACE analysis (FIG. 2C). Transcript A lacks thesequence that corresponds to exons 9 and 10 of PTCH1 (preliminarycomparisons of the intron exon junctions of PTCH2 with PTCH1 indicate asimilar genomic organization), with the open reading frame beingretained at the exon 8 to exon 11 junction. Exons 9 and 10 code for thelast part of the first extracellular loop and for transmembrane domains2 and 3 in the putative structure of the PTCH1 protein. Furthermore thistranscript also lacks a 5′ segment of the canonical exon 2, due to theuse of an alternative 3′ splice site present in this exon, with the openreading frame being maintained. The functional consequence of thisalternative splicing is not yet known, but it is interesting to notethat the extracellular loops in PTCH1 are presumed to be involved inbinding of the ligand SHH (Marigo et al., (1996), Nature 384, supra;Stone et al., (1996), Nature 384, supra) and that insertion of aneo-cassette in intron 9 of of the mouse PTCH1 gene is associated with asevere phenotype (Hahn, H., Wojnowski, L., Zimmer, A. M., Hall, J.,Miller, G. and Zimmer, A. (1998): “Rhabdomyosarcomas and radiationhypersensitivity in a mouse model of Gorlin syndrome”, Nature Med. 4,619-622). Furthermore, exons 9 and 10 encode part of a putative sterolsensing domain (Osborne, T. F. and Rosenfeld, J. M. (1998): “Relatedmembrane domains in proteins of sterol sensing and cell signallingprovide a glimpse of treasures still buried within the dynamic realm ofintracellular metabolic regulation”, Curr. Opin. Lipidol. 9, 137-140,also found in PTCH1, and which has recently been implicated in mediatingthe potent modulating effect of cholesterol on SHH/PTCH signalling(Cooper, M. K, Porter, J. A., Young, K. E., and Beachy, P. A. (1998):“Teratogen-mediated inhibition of target tissue response to Shhsignalling”, Science 280, 1603-1607). Thus, if PTCH2 also serves as areceptor for SHH and/or related factors, the receptor form lacking exons9 and 10 may show altered signalling properties. Transcript B containsadditional sequences between canonical exons 1 and 2, that originatefrom the 5′ end of intron 1. The open reading frame that includes theinitiator metionine of exon 1 is not maintained in this transcript,suggesting that, if this transcript is functional, either the methioninein exon 2 or non-methionine codons are used in order to produce aprotein product, in similarity to what has been proposed for thealternative spliced products of human PTCH1 (Hahn et al., Cell 85,supra). By radiation hybrid mapping the PTCH2 gene was localized to theshort arm of chromosome 1, in difference to PTCH1 residing on chromosome9q22.3.

The mouse and zebrafish homologs of PTCH2 have been reported to beexpressed in a partly overlapping pattern with PTCH1 during embryonicdevelopment and to be induced by SHH (Motoyama et al., (1998) NatureGenet. 18, supra, Concordet, J. P., Lewis, K. E., Moore, J. W.,Goodrich, L. V., Johnson, R. L., Scott, M. P., and Ingham, P. W. (1996):“Spatial regulation of a zebrafish patched homologue reflects the rolesof sonic hedgehog and protein kinase A in a neural tube and somitepatterning”, Development 122, 2835-2846), implicating a role in thissignalling pathway. We were with this background interested to analyzethe expression of PTCH2 in BCCs which show consistent upregulation ofPTCH1 in all tumor cells (Undén et al., (1997) Cancer res. 57, supra).In situ hybridisation was performed on six familial and four sporadicBCCs of different histological subtypes. A strong positive signal forPTCH2 mRNA was observed exclusively in the tumor cells of all BCCs.Notably, the signal was consistently stronger in the palisadingperipheral cells of the tumor nests (FIG. 2). These cells also showed apositive immunostaining for the cell proliferation marker, Ki-67.

The finding that in BCCs having frequent mutations in the PTCH1 gene,the expression of the PTCH2 mRNAs is upregulated, tightly links thenovel PTCH2 according to the invention with the PTCH/SHH cascade ofsignalling events. It is therefore likely that PTCH2 represents a targetgene of this pathway which is under the negative regulation of PTCH1,precisely as PTCH1 itself Moreover this observation strongly suggeststhat PTCH2 has functions distinct from PTCH1 since upregulation of PTCH2expression appears unable to compensate for inactive PTCH1 protein. Thisconclusion is also supported by the early embryonic lethality seen inPTCH1 (−/−) mice 5,13) and the lack of genetic heterogeneity in Gorlinsyndrome. However, whether PTCH2 may block the constitutive signallingof SMO, or could act as an additional SHH receptor, possible dependenton alternative splicing, remains as the subject of furtherexperimentation.

1. An isolated nucleic acid consisting of the sequence of SEQ ID NO: 5.2. An isolated nucleic acid encoding a protein consisting of SEQ IDNO:
 1. 3. A composition, comprising the nucleic acid according to claim1 or claim
 2. 4. A vector comprising a nucleic acid according to claim 1or claim
 2. 5. An isolated recombinant cell comprising a vectoraccording to claim
 4. 6. A kit for the detection of a human PTCH2 genecomprising in a container a nucleic acid according to claim 1 or claim2.